The steroid progesterone has an important role in the female reproductive system. Researchers interested in studying membrane progestin receptors (MPRS) developed a method to produce and purify the protein in active form. First, the researchers devised a way to prepare a specific MPR known as hMPRA using the machinery of yeast cells. In order to facilitate purification and identification in later studies, they manipulated the yeast cells so that they attached two different tags to the C-terminal end of the protein. The first tag, Compound 1, is a peptide sequence that acts as an epitope, part of a much larger peptide sequence that is recognized by the immune system. Glu-Gin-Lys-Leu-lle-Ser-Glu-Glu-Asp-Leu Compound 1
The steroid progesterone has an important role in the female reproductive system. Researchers interested in studying membrane progestin receptors (MPRS) developed a method to produce and purify the protein in active form. First, the researchers devised a way to prepare a specific MPR known as hMPRA using the machinery of yeast cells. In order to facilitate purification and identification in later studies, they manipulated the yeast cells so that they attached two different tags to the C-terminal end of the protein. The first tag, Compound 1, is a peptide sequence that acts as an epitope, part of a much larger peptide sequence that is recognized by the immune system. Glu-Gin-Lys-Leu-lle-Ser-Glu-Glu-Asp-Leu Compound 1
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
Related questions
Question
![The steroid progesterone has an important role in the female
reproductive system. Researchers interested in studying membrane
progestin receptors (MPRS) developed a method to produce and purify
the protein in active form.
First, the researchers devised a way to prepare a specific MPR
known as hMPRA using the machinery of yeast cells. In order to
facilitate purification and identification in later studies, they
manipulated the yeast cells so that they attached two different tags to
the C-terminal end of the protein.
The first tag, Compound 1, is a peptide sequence that acts as an
epitope, part of a much larger peptide sequence that is recognized by
the immune system.
The second sequence consisted of six consecutive histidine
residues (His). This sequence binds tightly to Ni2+ cations. In
chromatography, (His), tag labeled proteins can be eluted from Ni²+.
supported columns by adding a small molecule to the eluent that
mimics the side chain of histidine.
Glu-Gin-Lys-Leu-lle-Ser-Glu-Glu-Asp-Leu
Compound 1
After preparing hMPRA, the researchers conducted a binding assay
that utilized tritium labeled 1,2,6,7-[H³]-progesterone to measure the Ka
of the receptor while still in the membranes of the yeast cells.
hMPRA was then extracted from the membranes using
n-decyl-B-D-maltopyranoside, Compound 2. The researchers purified the
hMPRA with two successive rounds of chromatography that exploited
each of the tags. The buffers used to elute the protein contained
300 mM NaCl, 50 mM NaH₂PO, (pK, = 7.2), and various amounts of
NaOH (MM 40 g/mol). During the first chromatography step, a
specific chemical agent was immobilized on the stationary phase to
bind to the Compound 1 tag. After the second chromatography step,
which utilized the (His), tag, the researchers used the same binding
assay and found that K, was similar.
Adapted from M. B. Hossain, T. Oshima, S. Hirose, J. Wang, and T. Tokumoto, "Expression and Purification
of Human Membrane Progestin Receptor a (mPRA)." PLOS One. ©2015 Creative Commons Attribution.
The second purification step is which type of chromatographic
separation?
A. Affinity
OB. Size exclusion
O c. Cation exchange
O D. Anion exchange
The structure of Compound 2 is shown.
но.
HO
HO
OH
O
ОН
"OH
O A. 7.5 x 10-3
OB. 3.0 x 10-3
O c. 3.0 x 10²
O D. 1.5 x10-¹
OH
Compound 2
What structural feature(s) is(are) most important to the functioning of
this compound as described in the passage?
O A. Specific configuration of numerous chirality centers
OB. Multiple hydrolysable linkages
C. Combination of large hydrophobic and hydrophilic regions
O D. Presence of a reducing sugar
How many moles of NaCl were contained in 500 mL of the buffer
solution used to elute hMPRa?
Which experimental evidence suggests that the purified hMPRa
obtained by the researchers was in its native state?
The hMPRA that was obtained:
O A. retained both the Compound 1 and (His), tags.
OB. was purified by two separate chromatography steps.
O c. exhibited a binding affinity for progesterone that was similar
to that exhibited by native hMPRA.
OD. had a nearly identical molecular weight to hMPRA obtained
elsewhere.
The ligand of hMPRA is derived from which compound?
O A. Glucose
O B. Phenylalanine
O c. Glycerol
D. Cholesterol](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F167d28bb-887c-4334-a68e-3f4b17d619e7%2F17bea559-bbd7-40f6-94db-fddcb0809338%2Fky64vuq_processed.jpeg&w=3840&q=75)
Transcribed Image Text:The steroid progesterone has an important role in the female
reproductive system. Researchers interested in studying membrane
progestin receptors (MPRS) developed a method to produce and purify
the protein in active form.
First, the researchers devised a way to prepare a specific MPR
known as hMPRA using the machinery of yeast cells. In order to
facilitate purification and identification in later studies, they
manipulated the yeast cells so that they attached two different tags to
the C-terminal end of the protein.
The first tag, Compound 1, is a peptide sequence that acts as an
epitope, part of a much larger peptide sequence that is recognized by
the immune system.
The second sequence consisted of six consecutive histidine
residues (His). This sequence binds tightly to Ni2+ cations. In
chromatography, (His), tag labeled proteins can be eluted from Ni²+.
supported columns by adding a small molecule to the eluent that
mimics the side chain of histidine.
Glu-Gin-Lys-Leu-lle-Ser-Glu-Glu-Asp-Leu
Compound 1
After preparing hMPRA, the researchers conducted a binding assay
that utilized tritium labeled 1,2,6,7-[H³]-progesterone to measure the Ka
of the receptor while still in the membranes of the yeast cells.
hMPRA was then extracted from the membranes using
n-decyl-B-D-maltopyranoside, Compound 2. The researchers purified the
hMPRA with two successive rounds of chromatography that exploited
each of the tags. The buffers used to elute the protein contained
300 mM NaCl, 50 mM NaH₂PO, (pK, = 7.2), and various amounts of
NaOH (MM 40 g/mol). During the first chromatography step, a
specific chemical agent was immobilized on the stationary phase to
bind to the Compound 1 tag. After the second chromatography step,
which utilized the (His), tag, the researchers used the same binding
assay and found that K, was similar.
Adapted from M. B. Hossain, T. Oshima, S. Hirose, J. Wang, and T. Tokumoto, "Expression and Purification
of Human Membrane Progestin Receptor a (mPRA)." PLOS One. ©2015 Creative Commons Attribution.
The second purification step is which type of chromatographic
separation?
A. Affinity
OB. Size exclusion
O c. Cation exchange
O D. Anion exchange
The structure of Compound 2 is shown.
но.
HO
HO
OH
O
ОН
"OH
O A. 7.5 x 10-3
OB. 3.0 x 10-3
O c. 3.0 x 10²
O D. 1.5 x10-¹
OH
Compound 2
What structural feature(s) is(are) most important to the functioning of
this compound as described in the passage?
O A. Specific configuration of numerous chirality centers
OB. Multiple hydrolysable linkages
C. Combination of large hydrophobic and hydrophilic regions
O D. Presence of a reducing sugar
How many moles of NaCl were contained in 500 mL of the buffer
solution used to elute hMPRa?
Which experimental evidence suggests that the purified hMPRa
obtained by the researchers was in its native state?
The hMPRA that was obtained:
O A. retained both the Compound 1 and (His), tags.
OB. was purified by two separate chromatography steps.
O c. exhibited a binding affinity for progesterone that was similar
to that exhibited by native hMPRA.
OD. had a nearly identical molecular weight to hMPRA obtained
elsewhere.
The ligand of hMPRA is derived from which compound?
O A. Glucose
O B. Phenylalanine
O c. Glycerol
D. Cholesterol
Expert Solution
This question has been solved!
Explore an expertly crafted, step-by-step solution for a thorough understanding of key concepts.
This is a popular solution!
Trending now
This is a popular solution!
Step by step
Solved in 3 steps with 1 images
Recommended textbooks for you
Biochemistry
Biochemistry
ISBN:
9781319114671
Author:
Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:
W. H. Freeman
Lehninger Principles of Biochemistry
Biochemistry
ISBN:
9781464126116
Author:
David L. Nelson, Michael M. Cox
Publisher:
W. H. Freeman
Fundamentals of Biochemistry: Life at the Molecul…
Biochemistry
ISBN:
9781118918401
Author:
Donald Voet, Judith G. Voet, Charlotte W. Pratt
Publisher:
WILEY
Biochemistry
Biochemistry
ISBN:
9781319114671
Author:
Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:
W. H. Freeman
Lehninger Principles of Biochemistry
Biochemistry
ISBN:
9781464126116
Author:
David L. Nelson, Michael M. Cox
Publisher:
W. H. Freeman
Fundamentals of Biochemistry: Life at the Molecul…
Biochemistry
ISBN:
9781118918401
Author:
Donald Voet, Judith G. Voet, Charlotte W. Pratt
Publisher:
WILEY
Biochemistry
Biochemistry
ISBN:
9781305961135
Author:
Mary K. Campbell, Shawn O. Farrell, Owen M. McDougal
Publisher:
Cengage Learning
Biochemistry
Biochemistry
ISBN:
9781305577206
Author:
Reginald H. Garrett, Charles M. Grisham
Publisher:
Cengage Learning
Fundamentals of General, Organic, and Biological …
Biochemistry
ISBN:
9780134015187
Author:
John E. McMurry, David S. Ballantine, Carl A. Hoeger, Virginia E. Peterson
Publisher:
PEARSON